In approximately 20% of children and 65% of adults with acute lymphoblastic leukemia (ALL), leukemic cells persist despite intensive chemotherapy, leading to often fatal relapse. The hypothesis underlying the research proposed is that immune cells, i.e., T lymphocytes and natural killer (NK)cells, redirected by genetically-engineered ("chimeric") antigen receptors can eradicate drug-resistant ALL. In preliminary studies, receptors that recognize CD19 (a molecule highly expressed in ALL cells and absent in all normal cells except B lymphocytes) deliver stimulatory signals to immune cells resulting in powerful cytotoxicity against CD19+ALL cells. Specific Aim 1 is to identify stimulatory signaling molecules that induce maximum expansion and anti-CD19 cytotoxicity in NK cells. These studies stem from T.he observation that expression of anti-CD19 receptors in NK cells bypasses inhibitory mechanisms and confers anti-ALL cytotoxicity, and rely on a novel method to efficiently transduce the receptors in NK cells. The results should lead to clinical studies of NK cells in patients with refractory ALL and may expand the clinical use of these cells in cancer therapy. Studies in Specific Aim 2 will determine whether immune cells expressing anti-CD19 receptors can eradicate ALL in xenogeneic murine models of leukemia. The relative anti-leukemic capacity of NK cells and T cells, the potential benefits of 4-IBB and CD28 co-stimulation, and the effectiveness of infusing cells directed against two different leukemia-associated antigens will be assessed. If promising, the results should provide a strong rationale for clinical testing of receptor-modified autologous and allogeneic immune cells in patients with drug-resistant ALL. Specific Aim 3 is to increase the clinical safety of receptor-modified immune cells. Gene constructs that allow simultaneous expression of the receptors and of CD20 will be developed in efforts to render transduced cells susceptible to cytotoxicity mediated by Rituximab, an anti-CD20 antibody used clinically. The function of T and NK cells transduced with these constructs and their sensitivity to Rituximab will be tested in vitro and in vivo. Chimeric receptor-directed immunotherapy is an emerging area of cancer research. The research proposed should not only spur clinical studies of immune cells in patients with refractory ALL but also facilitate the development of effective cell therapies for other neoplasms.